VA scientists have discovered antiparasitic quinolones that are highly effective against malaria and toxoplasmosis. Malaria is a potentially fatal infection that is a deployment hazard for veterans. The many veterans who have HIV or who undergo hematopoietic stem cell or solid organ transplant are at risk of developing Toxoplasma encephalitis when they are immunosuppressed. Current treatments for malaria and prophylaxis for malaria are limited by the spread of drug resistance. Medicines for toxoplasmosis have high rates of side effects that are more common and problematic in HIV and transplant patients. This proposal builds on research that has identified ELQ-422 as a lead antiparasitic quinolone prodrug that is effective in animal models of toxoplasmosis and malaria, and is not toxic in cellular assays and efficacy experiments. The next step in preclinical testing is drug safety testing in animals. To achieve this, the maximum tolerated dose and no observed adverse effect level of ELQ-422 will be determined in rats. Demonstrating an acceptable therapeutic index of ELQ-422 in rats will allow for large animal testing and subsequent clinical trials. ELQ-422 is anticipated to be safe; however, identifying alternate antiparasitc quinolones is necessary in the event that ELQ-422 does not have an acceptable therapeutic index. The alkoxy carbonate ester promoiety of ELQ-422 that increases ELQ bioavailability close to 4 times will be applied to ELQs that were potent but limited by bioavailability. The promoiety will also be applied to a series of ELQ derivatives that will be synthesized to optimize a recently discovered ELQ that is 100 times more potent than ELQ-316, which is the active component of ELQ-422. These new prodrugs will be tested in a cascade of cellular and enzyme assays for efficacy and toxicity followed by in vivo studies of efficacy and pharmacokinetics. Lead compounds from these experiments will be evaluated for safety in rats in the same manner as ELQ-422. Finally, ELQ-422 and its active component ELQ-316 have been found to be synergistic with atovaquone (ATV) in acute models of toxoplasmosis and limit the development of drug-resistance in an animal model of human babesiosis, an infection that is similar to malaria. The underlying mechanism of synergy will be examined by comparing the cytocidal concentration and the concentration dependent kinetics of parasite inhibition of ELQ+ATV compared to each compound alone. In addition, the pharamacokinetic effects of ELQs and ATV on each other will be determined. The synergistic effect of ELQ+ATV will be tested in vivo as malaria prophylaxis and against latent Toxoplasma gondii infection. Overall, the proposed research will advance new broad-spectrum antiparasitic quinolones toward clinical evaluation and define treatment strategies that will enhance the clinical applicability of antiparasitic quinolones.